1. Field of the Invention
The present invention is in the fields of molecular biology and gene therapy. In particular, the invention provides isolated nucleic acid molecules encoding canine factor VIII and mutants, fragments or derivatives thereof. The invention also provides canine factor VIII polypeptides encoded by such isolated nucleic acid molecules, antibodies binding to such polypeptides, genetic constructs comprising such nucleic acid molecules, prokaryotic or eukaryotic host cells or whole animals comprising such genetic constructs, and methods and compositions for use in diagnosing disorders characterized by factor VIII deficiency, and therapeutic methods for treating diseases characterized by factor VIII deficiency.
2. Related Art
Overview
Factor VIII of the blood coagulation cascade is a trace plasma glycoprotein that participates as an essential co-factor in the middle phase of the intrinsic pathway of hemostasis. In humans, the protein is synthesized predominantly in hepatocytes although expression of the protein has also been documented in kidney, spleen and lymphoid tissue (Wion, K. D., et al., Nature 317:726-728 (1985)). In plasma, factor VIII circulates in a bimolecular complex with the multimeric protein von Willebrand factor (Hoyer, L. W.,Blood 58:1-13 (1981); Fay, P. J., and Smudzin, T. M., J. Biol. Chem. 265:6197-6202 (1990)), which protects it from proteolytic degradation by the natural anticoagulant factor, Protein C (Fay, P. J., and Walker, F. J., Biochim. Biophys. Acta 994:142-148 (1989)). Mutations in the gene that encodes factor VIII result in the X-linked inherited bleeding disorder, hemophilia A (Classic Hemophilia) (Hoyer, L. W., New Engl. J. Med. 330:38-47 (1994)).
The Human Factor VIII Protein
As alluded to above, factor VIII is synthesized in a variety of cellular sites including the liver, spleen and kidney (Wion, K. D., et al., Nature 317:726-728 (1985)). The most dramatic evidence of the involvement of hepatocytes in this process is the documentation of cures of hemophilic bleeding following liver transplantation (Lewis, J. H., et al., N. Engl. J. Med. 312:1189-1190 (1985)). In addition, evidence of factor VIII reconstitution following the transplantation of normal spleens into hemophilic dogs indicates that cells within this organ also play an important role in factor VIII synthesis (Webster, W. P., et al., N.C. Med. J. 28:505-507 (1967); Norman, J. C., et al., Surgery 64:1-16 (1968)).
In humans, factor VIII circulates in the plasma as a series of N-terminal heavy chain/C-terminal light chain heterodimers that are linked by a cationic bridge. The molecular weight of these complexes varies between .about.300 kD and .about.200 kD as a result of varying degrees of proteolysis of the central B domain of the protein.
The primary translation product encoded by the human factor VIII gene is a 2351 amino acid protein with a typical 19 residue N-terminal signal sequence (Wood, W. I., et al., Nature 312:330-336 (1984)). The sequence of the 2332 residue secreted human protein (SEQ ID NO:3) comprises three large tandem repeats of .about.350 amino acids (domains A1, A2 and A3) (Fass, D. N., et al., Proc. Natl. Acad. Sci. USA 82:1688-1691 (1985)). The amino terminal A1 and A2 domains are separated from the C-terminal A3 domain by the 980-residue B domain. Recombinant human factor VIII molecules from which the B domain has been deleted maintain full factor VIII cofactor activity and circulate in plasma with a normal half-life (Pittman, D. D., et al., Blood 81:2925-2935 (1993); Lind, P., et al., Eur. J. Biochem. 232:19-27 (1995)).
In plasma, human factor VIII is activated through the cleavage of two Arg-Ser bonds (Arg 372-Ser 373 in the N-terminal heavy chain and Arg 1689-Ser 1690 in the light chain) by the serine protease thrombin (Eaton, D., et al, Biochemistry 25:1986-1990 (1986); Pittman, D. D., and Kaufman, R. J., Proc. Natl. Acad. Sci. USA 85:2429-2433 (1988)). These same cleavages can be effected by activated factor X. A third peptide bond (Arg 740-Ser 741) is cleaved to release the B domain of the protein. Inactivation of human factor VIII cofactor activity is achieved by activated Protein C cleavage at Arg 336-Met 337 (Fay P. J., and Walker, F. J., Biochim. Biophys. Acta 994:142-148 (1989); Eaton, D., et al., Biochemistry 25:1986-1990 (1986)).
The other area of human factor VIII structure/function that has been explored extensively, relates to its interaction with von Willebrand factor (VWF). The binding site for VWF on human factor VIII is on the factor VIII light chain between residues Val 1670-Glu 1684 (Foster, P. A., et al., J. Biol. Chem. 263:5230-5234 (1988)). Post-translational sulfation of Tyr 1680 is critical to this process (Pittman, D. D., et al, Biochemistry 31:3315-3325 (1992)), and thrombin- or factor Xa-induced cleavage of Arg 1689-Ser 1690 will release VWF from human factor VIII.
The Human Factor VIII Gene
The gene that encodes human factor VIII is located on the long arm of the X chromosome close to the telomere, at cytogenetic band Xq28. The gene was cloned and characterized in 1984 by groups from the two American biotechnology companies Genentech and Genetics Institute (Gitschier, J., et al., Nature 312:326-330 (1984); Toole, J. J., Nature 312:342-347 (1984)). The human gene spans 186 kilobases of DNA and comprises 26 exons ranging in size from 69 basepairs (exon 5) to 3.1 kbp (exon 14). All the invariant splece donor and acceptor splice sites conform to the 5'GT/AG 3' rule, and remaining splice consensus sequences are in general agreement with other reported nucleotide frequencies.
THe human gene has 171 nucleotides (nts) of 5' untranslated sequence and 1,805 nucleotides of 3' UTR. In the 5' upstream region, a GATAAA sequence at nt -30 from the transcriptional start site likely represents an alternative TATA element. Preliminary studies of the human factor VIII promoter indicate that there are at least 12 cis-acting elements in the 1 kb of sequence upstream of the mRNA start site (Figueiredo, M. S., and Brownlee, G. G., J. Biol. Chem. 270:11828-11838 (1995); McGlynn, L. K., et al., Mol. Cell. Biol. 16:1936-1942 (1996)).
Molecular Genetic Pathology of Human Factor VIII
Two types of sequence changes have been found in the human factor VIII gene: neutral polymorphic changes, and mutations that result in functional factor VIII deficiency.
Human Factor VIII Polymorphisms
To date, nine nucleotide polymorphisms have been documented within or adjacent to the human factor VIII sequence (Peake, I., Thromb. Haemost. 67:277-280 (1992); Peake, I. R., et al., Bull. World Health Org. 71:429-458 (1993)). Seven of these sequence changes represent single nucleotide alterations demonstrable either by changes in restriction fragment length patterns or by allele specific oligonucleotide hybridization. The remaining two polymorphisms in introns 13 and 22 are examples of CA microsatellite repeats that demonstrate heterozygosity in &gt;70% of individuals (Lalloz, M. R. A., et al., Lancet 338:207-211 (1991); Windsor, S., et al., Br. J. Haematol. 86:810-815 (1994)). The analysis of human factor VIII polymorphisms continues to represent an important component of genetic studies for carrier testing and prenatal diagnosis in families in which hemophilia A is segregating (Peake, I. R., et al., Bull. World Health Org. 71:429-458 (1993)).
Human Factor VIII Mutations
Mutations within the human factor VIII gene give rise to the X-linked bleeding disorder hemophilia A. This disease has a population incidence of .about.1 in 10,000 males and represents the most common severe inherited bleeding disorder known in humans (Hoyer, L. W., New Engl. J. Med. 330:38-47 (1994)).
To date over 300 different human factor VIII mutations have been documented in patients with hemophilia A. As with other diseases in which widespread interest has been generated in molecular genetic pathology, a worldwide hemophilia A mutation database has been established to which all new human mutations are submitted (Tuddenham, E. G., et al., Nucl. Acids Res. 22:3511-3533 (1994)). A review of this database (available via Internet at http://146.179.66.63/usr/www/WebPages/main.dir/main.htm) shows that the majority of the mutations causing this disease are single nucleotide substitutions (total of 183 unique changes) distributed throughout the human factor VIII coding sequence. Many different gene deletions (117) and insertions (12) have also been documented.
Factor VIII in Other Species
Interest in the factor VIII molecule in species other than humans relates to two phenomena. First, spontaneously occurring hemophilia A has been identified in several other species including the dog (Graham, J. B., et al., J. Exp. Med. 90:97-111 (1949); Stormorken, H., et al., Scand. J. Haematol. 2:174-178 (1965); Giles, A. R., et al., Blood 60:727 (1982)), sheep (Neuenschwander, S., et al., Thromb. Haemost 68:618-620 (1992)), and horse (Archer, R. K., Vet. Rec. 73:338-341 (1961); Archer, R. K., and Allen, B. V., Vet. Rec. 91:655-661 (1972)). Second, recent results have indicated great potential in studying human gene therapy strategies in an animal model.
Despite this interest in factor VIII in non-human animals, however, primary sequences of factor VIII are only available for two other species: the mouse (SEQ ID NO:4) (Elder, B., et al., Genomics 16:374-379 (1993)) and the pig (SEQ ID NO:5) (Toole, J. J., et al., Proc. Natl. Acad. Sci. USA 83:5939-5942 (1986)). The complete porcine cDNA sequence obtained from PCR amplification of factor VIII sequences from a pig spleen cDNA library has recently been reported Healey, J. F., et al., Blood 88:4209-4214 (1996)).
A genomic fragment encoding the entire exon 14 and part of exon 15 of porcine factor VIII has been isolated, and the amino acid sequence of this region of the protein deduced (Toole, J. J., et al., Proc. Natl. Acad. Sci. USA 83:5939-5942(1986)). These results showed a marked contrast between the level of amino acid sequence identity between the porcine and human sequences in the B domain (.about.50% sequence identity and the remainder of the protein (approaching 85% sequence identity). In addition, the porcine B domain shows deletions of over 200 amino acids when compared to the human protein.
The murine factor VIII cDNA sequence has also been reported (Elder, B., et al., Genomics 16:374-379 (1993)). The 6.9 kb murine factor VIII coding sequence shares 82% sequence identity with the coding sequence of human factor VIII. Outside of the sequence encoding the B domain the sequence identity at the nucleotide level is 88%. At the amino acid level, an overall identity of 74% is present and this figure increases to 87% when only residues outside of the B domain are considered. Amino acid sequence identity for the B domain is significantly lower at 55% for human versus mouse. The residues present at critical sites for factor VIII cleavage are conserved in the mouse sequence and sites for essential post-translational modification are also maintained. Finally, the shorter 7.2 kb factor VIII transcript derived from the mouse gene represents the consequence of a smaller 3' untranslated region in the mouse.
Since the identification of spontaneously occurring hemophilia A in dogs (Graham, J. B., et al., J. Exp. Med. 90:97-111 (1949); Stormorken, H., et al., Scand J. Haematol. 2:174-178 (1995); Giles, A. R., et as., Blood 60:727 (1982), there has been increasing interest in the use of canines as model systems for the study of the physiology of human diseases characterized by factor VIII deficiencies (e.g., hemophilia A). Furthermore, the canine has shown promise as a model system for the development of methods of detecting and treating such diseases in humans.
It would therefore be advantageous to provide compositions comprising isolated nucleic acid molecules encoding canine factor VIII or polypeptides encoded by such nucleic acid molecules, and diagnostic and therapeutic methods for detecting and treating factor VIII deficiencies (spontaneous or induced) in dogs. These methods and compositions would be particularly useful in modeling similar approaches to diagnosis and treatment of human factor VIII deficiencies. The present invention therefore provides such methods and compositions.